There are currently ~100,000 patients on the organ transplant waiting list in the US, a number that far exceeds the supply of available organs, and that continues to grow ~5% each year. The most promising solutions, bioartificial tissue and organ construction and donor organ reengineering methodologies, are both ultimately limited by biopreservation technologies, as any tissue engineered products prepared in a laboratory will have to be stored for a period of time until utilization. The current gold standard for whole organ preservation is cold storage on ice for up to 72 hours, during which time the organ continuously deteriorates. A superior biopreservation method that extends the tissue storage time beyond current limitations is yet to be developed. Such a method would provide a crucial enabling technology for tissue and organ preservation, tissue and organ transport, and tissue and organ transplantation. The objective of this study is to extend the viable preservation time of hepatic tissues by sub-zero non- freezing (SZNF) storage in a supercooled preservation medium. The central hypothesis of this study relies on two phenomena: 1) that 3-O-methyl-glucose (3OMG) lowers achievable stable SZNF temperature without major toxic side effects, and that 2) rewarming by normothermic perfusion reduces reperfusion damage. Our hypothesis has been formulated based on our preliminary findings establishing 3OMG as a minimally toxic cryoprotectant for hepatocytes, and establishing that normothermic perfusion can significantly reverse the damaging effects of ischemia. The rationale of the study is that if supercooled preservation can be achieved while avoiding antifreeze toxicity, then organ metabolism can be further slowed thereby reducing anoxic/ischemic damage to minimal levels. Establishment of a sub-zero nonfreezing preservation technology will be a welcome innovation to the field. The work described herein will help develop this enabling technology of supercooled storage, and also establish quantitative standards for evaluating the liver and bioartificial organ viability following preservation. While we focus on the liver, we expect that the protocols established here will also serve as the basis for subzero nonfreezing preservation of other tissue engineered products, such as artificial organ substitutes and seeded scaffold constructs. Public Health Relevance Statement (provided by applicant): There are currently 97,000 patients on the transplant waiting list, and the number increases by ~5% every year. A critical bottleneck in making more donor organs as well tissue engineering alternatives available to the public is the limited preservation duration. The objective of this study is to extend the viable preservation time of organs and bioartificial alternatives by enabling extended storage at sub-freezing temperatures without ice formation. The results of this study are expected to directly improve public health by increasing donor organ availability and making more transplantations possible.